GeneJET™ Plasmid Miniprep Kit

- Store at room temperature

Catalog#	Size, concentration	Certificate of Analysis	MSDS

K0502	for 50 preps.	K0502
K0503	for 250 preps.	K0503

Product information

Overview of purification
procedure

Features

Efficient – high yields of up to 20 µg of high quality plasmid DNA.
Fast – procedure takes less than 14 min.
Convenient – no phenol/chloroform extraction or alcohol precipitation required.
Pure – purified DNA is immediately ready to use.

Applications

Fast isolation of high purity plasmid DNA suitable for all conventional molecular biology procedures, including:
– FastDigest® or conventional restriction digestion,
– automated fluorescent and radioactive sequencing,
– PCR,
– in vitro transcription,
– transformation,
– transfection of robust cell lines.

Description

The GeneJET™ Plasmid Miniprep Kit is a simple, rapid and cost-effective system for the isolation of high quality plasmid DNA from recombinant E.coli cultures. The kit utilizes an exclusive silica-based membrane technology in the form of a convenient spin column. The kit recovers up to 20 µg of high copy plasmid DNA per isolation procedure.

Principle

A bacterial culture is harvested and lysed. The lysate is then cleared by centrifugation and applied on the silica column to selectively bind DNA molecules at a high salt concentration. The adsorbed DNA is washed to remove contaminants, and the pure plasmid DNA is eluted in a small volume of elution buffer or water. The purified DNA is ready for immediate use in all molecular biology procedures such as fast and conventional digestion with restriction enzymes, PCR, in vitro transcription, transformation and automated sequencing.

Quality Control

The kit is tested for the isolation of pUC19 DNA from E.coli according to the protocol. The quality of the isolated DNA is evaluated by spectrophotometric assay, agarose gel electrophoresis, digestion with FastDigest® restriction enzymes and automated sequencing.

Components

Resuspension Solution
Lysis Solution
Neutralization Solution
Wash Solution (concentrated)
RNase A Solution
Elution Buffer
Spin Columns
Collection Tubes
Detailed Protocol

Figure 1. Fast clone analysis.
A 2.3 kb PCR fragment was cloned into pUC19 vector. Plasmid DNA from overnight bacterial cultures of recombinant clones was purified using the GeneJET™ Plasmid Miniprep Kit and analyzed by double digestion with FastDigest™ EcoRI and FastDigest™ HindIII in 5 min at 37°C.
M₁, M₂ – ZipRuler™ Express DNA Ladder Set
C – control pUC19 DNA digested with FastDigest® EcoRI and HindIII
1-6 – miniprep DNA from recombinant clones, double digested with FastDigest® EcoRI and HindIII

Effect of time, amount of template on the specific radioactivity of a DNA probe

Figure 2. Electropherogram demonstrates the high quality sequencing data of plasmid DNA purified with GeneJET™ Plasmid Miniprep Kit.
Plasmid DNA was purified using the GeneJET™ Plasmid Miniprep Kit from E.coli and sequenced with a T7 sequencing primer. The sequencing reaction was analyzed on an ABI 3730 DNA Analyzer. More than 750 nucleotides can be read.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Analysis of Recombinant Clones

Analyze 4-6 white colonies for the presence and orientation of the DNA insert using one of the following methods.
Restriction analysis. Isolate plasmid DNA from an overnight bacterial culture using a convenient plasmid miniprep method such as GeneJET™ Plasmid Miniprep Kit. Use FastDigest® restriction enzymes to digest DNA from recombinant clones in just 5 min.
Sequencing. Isolate plasmid DNA from an overnight bacterial culture using a reliable plasmid miniprep method such as GeneJET™ Plasmid Miniprep Kit. Sequence the insert using appropriate sequencing primers.
Colony PCR. Use the following protocol for colony screening by PCR.

Prepare enough PCR master mix for the number of colonies analyzed plus one extra. For each 20 µl of reaction, mix the following reagents:

Component	Taq DNA Polymerase or DreamTaq™ DNA Polymerase	PCR Master Mix (2X)
*10X Taq* buffer**	2 µl	-
dNTP Mix, 2mM each	2 µl	-
25 mM MgCl₂	1.2 µl	-
M13/pUC Reverse Sequencing primer (#SO101)	0.6 µl (10 µM)	0.6 µl (10 µM)
M13/pUC Forward Sequencing primer (#SO100)	0.6 µl (10 µM)	0.6 µl (10 µM)
Taq DNA polymerase	0.1 µl (0.5 u)	-
PCR Master Mix (2X)	-	10 µl
Water, nuclease-free	to 20 µl	to 20 µl
Total volume	20 µl	20 µl

Mix thoroughly, spin briefly and aliquot 20 µl of the mix into the PCR tubes on ice.
Pick up an individual colony with a sterile pipette tip and resuspend it in 20 µl of the PCR master mix. Make a short strike with the same tip over culture plate to save the clone for repropagation.
Perform PCR: 95°C, 3 min; 94°C, 30 s, 45°C*, 30 s, 72°C 1 min/kb; 30 cycles.
Analyze on a gel for the presence of the PCR product of the expected length.
* Depends on primer pair used (Tm-5).

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